The present invention relates to the design of transcriptional cascade circuits to amplify gene expression. It also relates to the use of these systems for the overproduction of polypeptides such as therapeutic proteins, enzymes, hormones, growth factors, and apoliproteins in vitro, and in cells, i.e., cell cultures. This has great industrial utility, e.g., in the biotechnology and pharmaceutical industries.
Overexpression of cloned genes is very convenient for production of either recombinant polypeptides or specific cell metabolites for basic research, and the pharmaceutical and biotechnology industries generally. Production of large amounts of cloned genes has traditionally been achieved by combining gene amplification with strong promoters regulated by repressors. However, these conventional strategies typically have the disadvantages that: 1) maintenance of plasmid expression vectors typically requires selection with antibiotics, giving rise to metabolic burden and additional costs for large scale industrial production (Nilson and Skogman 1986); 2) low level expression is indicated when dealing with toxic proteins, and in avoiding accumulation of mutations in the recombinant protein products themselves (Mertens et al., 1995; Vilette et al. 1995), which is difficult to achieve when the cloned gene is in multicopy due to the multiple copy number of traditional plasmid vectors and the high basal (xe2x80x9cleakyxe2x80x9d) level of expression of most traditionally used promoters, e.g., tac or trc; 3) traditional inducers, e.g., IPTG in lac expression systems, are expensive and have a certain degree of toxicity (Figge et al. 1988); 4) high expression of recombinant proteins has been shown to reduce host cell growth rate and, concomitantly, overall protein synthesis (Bentley et al., 1990; Dong et al. 1995), presumably due to increased metabolic burden on the host; and 5) a considerable number of existing expression systems only replicate in E. coli, which may limit expression of certain proteins, e.g., those desired to be secreted.
An alternative expression system that fulfills some of the above requirements uses miniTn5 transposon vectors (de Lorenzo and Timmis 1994) to insert heterologous genes into the bacterial chromosome, thereby allowing high stability of expression (Cebolla et al. 1993; Suarez et al. 1997). Suarez in particular describes the stable production of pertusic toxin in Bortedetella bronchiseptica by miniTn5-mediated chromosomal insertion and expression using a salicylate regulatory system. Salicylate is a benzoate inducer 1000-fold less expensive than IPTG (SIGMA catalog 1998). The system is based on the nahR regulatory gene, which encodes a positive regulator activated by salicylate and its target promoter Psal (de Lorenzo et al. 1993). However, expression levels obtained are relatively poor (0.1% of total proteins). This low level is likely because the genes are in monocopy in the chromosome.
If yield could be improved while maintaining the advantages of low basal levels, stability, broad host range, and low cost, the nahR/Psal regulatory system would have great industrial utility.
We have designed a cascade system that allows 10 to 20 fold greater expression over the standard nahR/Psal system while substantially retaining one or more of that system""s innate advantages. To achieve this, another regulatory element, xylS2 and its target promoter Pm, is coupled to Psal expression in a cascade circuit. The xylS2 regulatory gene responds to the common inducer and has more gene expression capacity than standard nahR/Psal. Synergistic activation of the Pm promoter by the XylS2 transcriptional activator can be achieved by simultaneously increasing the intracellular concentration and specific activity of activator/regulator in the presence of a common benzoate derivative inducer, e.g., salicylate.
Accordingly, in a first aspect the invention features a cascade genetic circuit comprising one or more nucleic acid constructs encoding a plurality of transcriptional regulators, said encoded regulators arranged in a hierarchical order such that expression of an upstream regulator from said plurality stimulates expression of a downstream regulator from said plurality; and a final target promoter, said final target promoter responsive in a dose-dependent fashion to a terminal downstream regulator of said plurality of regulators.
In certain preferred embodiments, it may be useful to introduce the final target promoter alone, e.g., via PCR, into a host genome at designated position to determine effect on expression of the downstream sequence. To do this, it may first be desirable to disable or knock out the native promoter, gene, or nucleic acid sequence. In other preferred embodiments, as described below, heterologous genes and sequences are preferred for use with the cascade circuit and, accordingly, may be introduced.
In one especially preferred embodiment, the cascade genetic circuit further comprises a multiple cloning site downstream of the final target promoter.
In another preferred embodiment, the cascade genetic circuit, or at least a portion thereof, is present as a chromosomal integration in a host cell. In a different, not necessarily mutually exclusive embodiment, at least one of said one or more nucleic acid constructs is present as an autoreplicative plasmid.
In a further embodiment, the cascade genetic circuit, or at least a portion thereof, is responsive to an inducer, preferably an inducer that is capable of inducing the expression of more than one regulator in the cascade. In preferred embodiments, the inducer is a benzoate derivative, preferably, although not necessarily, salicylate.
In another aspect, the invention features a cell, tissue, or organism comprising the cascade genetic circuit of any of the preceding claims. Preferably, the cell is selected from the group consisting of procaryotic and eukaryotic cells. As concerns eukaryotic cells, mammalian, insect, yeast, and plant cells are preferred. As concerns procaryotic cells, gram-negative bacterial cells are preferred.
In yet another aspect, the inventon features methods of regulating the expression of a nucleic acid sequence, comprising establishing a cascade genetic circuit according to any of the cascade genetic circuit embodiments described above; placing said nucleic acid sequence under control of said final target promoter; and inducing said cascade genetic circuit to stimulate expression of said nucleic acid sequence.
Preferably the nucleic acid sequence encodes a polypeptide selected from the group consisting of enzymes, hormones, growth factors, apolipoproteins, therapeutic proteins, diagnostic proteins, and portions or derivatives thereof. In other preferred embodiments, the nucleic acid sequence encodes an anti-sense molecule, ribozyme, rRNA, tRNA, snRNA, or simply a diagnostic RNA molecule. In certain preferred embodiments, the nucleic acid encodes a reporter gene product useful in diagnostics.
By using regulatory genes of the control circuits for the expression of catabolic operons, a cascade expression system for amplifying gene expression was constructed. The system is based on the activation characteristics of the Pm promoter by the XylS2 mutant transcriptional activator. Strength of Pm activation depends on both the amount of XylS2 protein and its specific activity, which is enhanced by the presence of salicylate and other benzoate derivatives. To couple the increase of XylS2 intrinsic activity and XylS2 intracellular concentration, the expression of xylS2 is under the control of the Psal promoter and the NahR transcriptional activator, that is also activated in response to common inducers. The synergistic action of both transcriptional regulators lead to 10 to 20-fold amplification of the gene expression capacity with regard to each individual expression system.
One embodiment of the system comprises a cassette having the regulatory genes nahR/Psal::xylS2 flanked by transposable sequences which facilitate stable insertion into the chromosome of a cell, e.g., a gram negative bacteria. A complementary expression module containing the target promoter Pm upstream of a multicloning site for facilitating the cloning of a recombinant DNA is used as part of the system. The expression module can be introduced in multicopy plasmid form or else transferred to the chromosome, e.g., via minitransposon delivery vectors (if either stability of the expression and/or the lowest basal level are desired). To achieve this, the expression module with the Pm promoter and the heterologous gene/s are preferably flanked by rare restriction sites, e.g., NotI, for further cloning utility.
An ideal expression system should be tighly regulated (i.e. to have very low basal level of expression and a high level of expression in the presence of inducer). For large scale fermentations, it is very convenient that the inducer be cheap and that the overexpression of the gene be stable, preferably without selective pressure. The capacity of the culture to reach high biomass should be affected as little as possible, and convenient for use in a broad range of organisms. Salicylate-induced expression using nahR/Psal in the chromosome of gram negative bacteria has proven to be very stable and tightly regulated (Suarez et al. 1996). However, the expression level obtained is very low due to single copy presence and limited gene expression capacity.
In contrast, the xylS/Pm expression system has shown to have an outstanding range of activity that depends both on the specific activity of the XylS transcriptional regulator and on the intercellular concentration of XylS; manipulation of either affects expression (Kessler et al. 1994).
The transcriptional activity of Pm in vivo appears to be non-saturable because, despite overexpression of XylS, expression from Pm continues in response to 3-methyl-benzoate inducer. However, the expression systems based on the xylS/Pm system maintain constant amounts of xylS expression, which waste the potential increment in gene expression capacity if xylS expression could be made inducible. As the Applicants demonstrate herein, this can be achieved by coupling the expression of xylS to another expression system (first system). If the signal that induces transcriptional regulator expression were the same that the one that activates it, the signal could produce synergy in the activation of gene expression from the Pm promoter.
The present invention describes this: synergistic signal amplification using coupled expression systems. To use regulators responding to a common signal, nahR/Psal was used as a first regulatory system and xylS2, a mutant of xylS able to respond to salicylate (Ramos et al. 1986), as a second regulatory system. A 1.2 Kb fragment with the xylS2 gene was cloned by digestion with HindIII and partial digestion with NcoI, and insertion in the same sites of pFH2 (Table 1). The resultant plasmid pNS2 was digested with NotI and the fragment with xylS2 was inserted into the plasmid pCNB4 (de Lorenzo et al. 1993). This regulator is left under the control of the nahR/Psal system and flanked by the insertion sequences of miniTn5. The resultant plasmid pCNB4-S2 (FIG. 2a) has a R6K replication origin (Kahn et al. 1979), that can only replicate in strains expressing xcfx80 protein. E. coli xcexpir lysogen can express that protein and thus, replicate the miniTn5 delivery plasmid (Herrero et al. 1990).
By using the donor strain of miniTn5 vectors S17-1(xcexpir ) one can transfer the regulatory cassette to other gram negative strains using standard biparental conjugation, and select for recipient bacteria using selective markers determined from the minitransposon (Herrero et al. 1990; de Lorenzo and Timmis 1994). To verify that it is an insertion by transposition and that the plasmid R6K has not been inserted, the transconjugant colonies are checked for the loss of xcex2-lactamase of the suicide plasmid. Every strain with the regulatory cassette nahR/Psal::xylS2 would produce the regulatory protein XylS2 in response to salicylate (FIG. 1).
This strain can then be used for insertion by conjugation or transformation of a second regulatory cassette that contains the Pm promoter fused to the heterologous gene of interest. One such construct, pTSPm, is a miniTn5 vector that contains within the insertion sequences a streptomycin resistance gene, the Pm promoter, and a NotI restriction site (FIG. 2a) to insert the heterologous gene.
To construct pTSPm, a fragment with the omega interposon (Fellay et al. 1987) was inserted in the BamHI site of the vector pUC18Sfi-KmR-xylS-Pm-Sfi (de Lorenzo et al. 1993) which removed a fragment with KmR and xylS. The resultant plasmid was digested with SfiI and the biggest fragment was cloned into the pUT backbone of a miniTn5 vector (Herrero et al. 1990) to obtain the plasmid pTSPm. Auxiliary vectors of Table 1 can be used to clone the heterologous genes and then subclone into the NotI site of pTSPm.
A second expression vector with a ColE1 replication origen (pCCD5) contains the Pm promoter upstream of a multicloning site, and a good translation initiation sequence for convenience in cloning heterologous genes (FIG. 2b). Plasmid pCCD5 was constructed cloning an ApoI fragment containing the rrnBT1 transcriptional terminator produced as a PCR fragment from pKK232-8 with the oligos 5xe2x80x2-GCAAATTTCCAGGCATCAAATAA-3xe2x80x2 (SEQ ID NO: 1) and 5xe2x80x2-GGGAATTCCCTGGCAGTTTATGG-3xe2x80x2 (SEQ ID NO: 2), into the EcoRI site of pFH2 (Table 1). Following ligation, a unique EcoRI site results and those plasmids with intact MCSs can be selected. The Pm promoter was obtained by PCR, using the oligos A-Pm (5xe2x80x2-GTGTCAAATTTGATAGGGATAAGTCC-3xe2x80x2 (SEQ ID NO: 3)) and Pm-E (5xe2x80x2-GCCTGAATTCAGGCATTGACGAAGGCA-3xe2x80x2 (SEQ ID NO: 4)) as primers, and pUC18Sfi-KmR-xylS-Pm-Sfi as template. Digestion with ApoI (underlined) of the amplified fragment (0.4 Kb) gave a fragment compatible with EcoRI termini, but only the extreme of the fragment downstream of the Pm promoter regenerated the EcoRI site in the junction. Therefore, introduction of the Pm ApoI fragment in the proper sense into the EcoRI linearized intermediate plasmids, rendered vectors that contained the Pm promoter preceeding an intact MCS, with all the key elements flanked by NotI sites.
This last feature may allow to clone in mini-Tn5 delivery vectors if monocopy or stability of the expression system are required. There exists up to 8 different makers in miniTn5 vectors with the NotI site where the Pm fusion may be cloned (de Lorenzo 1994). The resultant vectors may then be inserted into the chromosome of the receptor bacteria by conjugation. Alternatively, cloned genes in pCCD5 under Pm control may be transformed in strains with the regulatory cassette in the chromosome to overexpress the heterologous gene from the plasmid.